Chelex 100 resin solution

To obtain genomic DNA from environmental samples, the 46 single colonies were isolated using the single-cell isolation method and transferred to a droplet of DNA nuclease-free water (Bioneer Co., Daejeon, Korea). After taking pictures of each colony, they were washed three times with the DNA nuclease-free water and transferred to a 0.2 mL PCR tube containing 100 μL of 10% (w/v) Chelex^® 100 resin solution (Bio-Rad Laboratories, Hercules, CA, USA). Chelex-stored samples were kept from 1 to 7 d in the dark at 4°C until DNA extraction.

A total of 244 unrelated animals of both sexes were randomly sampled from six provinces representing all the continental agroecosystems of Ecuador: Bolívar, Chimborazo, Cotopaxi, Guayas, Morona Santiago, and Tungurahua (Supplementary Material: Figure S1, Table S1). Specific sampling and geospatial locations are reported in Table S1, according to the 26 municipalities within the 6 provinces of precedence. Up to 200 μL of EDTA K3 (BD Vacutainer, Franklin Lakes, NJ, USA) anticoagulated blood was loaded in the collection circle of Whatman^® FTA^® Cards (GE healthcare Life Science, Little Chalfont, Buckinghamshire, UK). The cards were dried at 25 °C for a minimum of 3 h and then stored in paper envelopes until use. DNA was extracted for both mitochondrial DNA and STRs (short tandem repeats) analysis, following a modification of the method described by Walsh et al. [14 (link)]. Briefly, three circles were cut in the spotted cards using a 2 mm Harris Micro punch (GE healthcare Life Science, Little Chalfont, Buckinghamshire, UK), which was cleaned using 1% bleach solution between each sample. The circles were placed in a PCR plate and incubated in 100 µL of a 5% CHELEX 100 resin solution (Bio-Rad, Hercules, CA, USA) at 95 °C for 15 min, 60 °C for 15 min, and finally 99 °C for 3 min. The lysate was removed and frozen at −20 °C until use.

DNA extraction was performed using 5% Chelex^® 100 Resin solution (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol. 200 μL of the sample was transferred to a new E-tube, centrifuged at 8000× g for 10 min, the supernatant was discarded, and 100 μL of 5% Chelex^® 100 Resin solution was added to the remaining cell pellet, followed by vortexing and spinning down. Then, samples were incubated at 100 °C for 10 min, centrifuged at 11,000× g for 10 min, and supernatant transferred to a new E-tube. The extracted genomic DNA was checked for concentration and purity using a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA) and stored at −18 °C before analysis.